Abstract This research proposal focuses on the secretory stage of amelogenesis where enamel mineral ribbons initiate on dentin mineral and elongate until the enamel layer reaches its final dimensions. Many genes cause inherited enamel malformations (amelogenesis imperfecta, AI). Defects in genes necessary for the secretory stage typically cause thinner (hypoplastic) enamel with a rough surface. Dental enamel forms in a defined extracellular space that is established and modified by ameloblasts. We cite 19 human AI genes causing enamel hypoplasia, but focus on those most directly associated with the extracellular enamel matrix. Five genes encode secreted matrix proteins: ENAM, AMBN, AMELX, MMP20, and ODAPH. Except for ODAPH (formerly C4orf26), these genes/proteins are well-characterized. There are 2 key secretory stage ion transporters: SLC4A4 (NCBe1; transports bicarbonate into enamel matrix) and SLC13A5. Bicarbonate is known to neutralize the acid generated by mineral deposition, but why absence of the citrate transporter (NaCT) encoded by SLC13A5 causes severe enamel malformations is unknown. SLC13A5 is required in soft (liver, brain) and hard (bone, teeth) tissues. Surprisingly ~80% of all citrate in the body is in bone. Citrate is part of the Citric Acid Cycle where citrate is generated from oxaloacetate, acetyl- coenzyme A, and water in a reaction catalyzed by citrate synthase within the mitochondrial matrix. Citrate likely plays 1 of 2 roles: It could be transported into ameloblasts (influx) across its proximal membrane to increase energy metabolism or be transported out of ameloblasts (efflux) across its distal membrane into the developing enamel matrix. Based upon findings of citrate in bone and enamel, we hypothesize citrate is secreted and helps to regulate enamel ribbon deposition. The critical roles of ODAPH and SLC13A5 in enamel ribbon formation represent two major gaps in our understanding of amelogenesis. We close these gaps by generating and validating Odaph and Slc13a5 knockout (KO) mice expressing premature stop codons homologous to human AI-causing mutations. We also generate a Slc13a5 knockin (KI) expressing 3 FLAG epitopes on its C-terminus. Two Specific Aims are proposed: SA1: To develop and validate a Slc13a5 KO mouse model homologous to human AI (p.Arg333*) and to generate a Slc13a5FLAG KI mouse for sensitive and specific immunolocalization (IHC). UG3: generate a Slc13a5-/- mouse AI model in C57BL/6J background using CRISPR/Cas9. UG3: generate a Slc13a5FLAG mouse wild-type-tagged model for sensitive and specific localization. UH3: validate the Slc13a5-/- mouse by characterizing its enamel. Validate Slc13a5FLAG mouse by IHC. SA2: To develop and validate an Odaph KO mouse model homologous to human AI (p.Cys43*). UG3: generate an Odaph-/- mouse AI model in C57BL/6J background using CRISPR/Cas9. UH3: validate the Odaph-/- mouse by characterizing its enamel phenotype.